This invention claims priority of a German patent application DE P 100 04 233.3 filed Feb. 1, 2000 which is incorporated by reference herein.
The invention relates to a confocal scanning microscope, having a light source for illuminating an object, which is to be investigated, with exciting light, at least two detection channels exhibiting detection light being produced.
Microscopes of the type mentioned at the beginning are known in practice and exist in the most varied embodiments. An example of such a microscope is formed by a confocal scanning microscope in the case of which an object to be investigated is scanned with the aid of a light beam or the exciting light. The microscope generally comprises a light source and a focusing optical system with the aid of which the light from the source is focused onto a pinhole stop. In this case, a beam splitter, a scanning device for beam control, a microscope optical system, a detection stop and detectors for detecting detection and/or fluorescent light are provided.
The illuminating light is mostly coupled in via the beam splitter. The focus of the light beam is moved with the aid of the scanning device in a sample plane. For this purpose, it is customary to use two mirrors which are tilted, the deflection axes mostly being perpendicular to one another, such that one mirror deflects in the X-direction and the other deflects in the Y-direction. Tilting of the mirrors is accomplished, for example, with the aid of galvanometer control elements. The fluorescent or reflected light coming from the sample passes in this mostly conventional descanning arrangement via the same scanning mirror back to the beam splitter and passes the latter in order subsequently to be focused onto the detection stop, downstream of which the detectors are located. Detection light which does not originate directly from the focusing region takes a different light path and does not pass the detection stop, and so point information is obtained which leads through scanning of the object to a three-dimensional image. Illumination and detection take place in this case on the objective side, that is to say on sides of the microscope optical system.
It is also possible in a transmitted-light arrangement for the fluorescent light or the transmitted lightxe2x80x94the transmission of the exciting lightxe2x80x94, for example, to be detected on the condenser side, that is to say on the side of a condenser arranged downstream of the object. The detection-light beam then does not pass via the scanning mirrors to the detector. Such an arrangement is denoted as a non-descanning arrangement.
In order to detect the fluorescent light, there would be a need in the transmitted-light arrangement for a condenser-side detection stop in orderxe2x80x94as in the descanning arrangement describedxe2x80x94to achieve a three-dimensional resolution. In the case of two-photon excitation, however, it is possible to dispense with a condenser-side detection stop, since the probability of excitation is a function of the square of the photon density or the intensity, which is naturally much higher at the focus than in the neighboring regions. The fluorescent light to be detected therefore originates with high probability in overwhelming proportion from the focusing region, and this renders superfluous further differentiation of fluorescence photons from the focusing region from fluorescence photons from the neighboring regions with the aid of a stop arrangement. However, the detection of the transmitted light and/or the condenser-side detection of the fluorescent light can be helpful in the case of single-photon excitation, as well.
Particularly against the background of a yield of fluorescence photons which is low in any case for two-photon excitation, a non-descanning arrangement in which less light is generally lost on the detection-light path is of interest.
It is known from EP 0 627 643 A1 to raise the signal yield by electronic addition of the signals of the descanning and non-descanning detectors. Two detection channels are thus superimposed electronically in this case.
In the known electronic addition or superimposition of the signals, it is a problem that this form of electronic superimposition of the detector signals is complicated and slow. In particular, the detectors used have to be set in a complicated fashion.
It is therefore the object of the present invention to provide a confocal scanning microscope which achieves a high signal yield and a high signal-to-noise ratio by the use of simple means.
The above object is achieved by a confocal microscope which comprises: a light source defining exciting light for illuminating an object, a scanning device, a first and second detection light being generated in the object wherein the detection light defines at least two detection channels, a superimposing device for optically superimposing the at least two detection channels and a detector assembly with at least one detector for detecting the detection light.
In a way according to the invention, it has firstly been realized that raising the signal yield can be achieved not solely by electronic addition of the signals of the detectors used. Also in a way according to the invention, optical superimposition of at least two detection channels by means of a superimposing device is provided for this purpose. With the microscope according to the invention, it is possible to dispense entirely with vulnerable electronic components which have additionally to be integrated in the known microscope. In this case, the complicated setting of the detectors used is also eliminated. Finally, the optical superimposition provides a quicker superimposition or addition technique than the conventional electronic addition technique.
Consequently, the microscope according to the invention realizes a microscope in which a high signal yield and a high signal-to-noise ratio is achieved by simple means.
In concrete terms, the detection light of at least two detection channels could be detected in a common detector assembly having at least one detector. However, it would also be possible to detect more than two detection channels in a common detector assembly.
The location of the superimposition of the detection light of the respective detection channels is not prescribed in principle. In a structurally particularly simple design, it would be possible, however, for the detection light of the detection channels to be optically superimposed in the detector assembly. In this case, the detection light of the respective detection channels is fed to the detector assembly in a suitable way.
The detector assembly could be a descanning detector assembly, it being rendered possible in a practical way to make use for optical superimposition of a descanning detector assembly frequently already present in a microscope. An additional detector assembly is therefore not required.
The detector assembly could, however, also be a non-descanning detector assembly. It would then be necessary when extending a conventional microscope which originally has a descanning detector assembly to provide a further detector assembly with the aid of which it is then possible to detect the detection light of all the detection channels. In particular, it would be possible thereby for the detection light of at least one detection channel to be guided to the detector assembly before it traversesxe2x80x94as originally plannedxe2x80x94a scanning device. For this purpose, the detection light could preferably be guided to the detector assembly by splitting between an objective and the scanning devicexe2x80x94from the original beam path. For splitting purposes, a color beam splitter could, in a particularly simple way, be arranged in a beam path of the microscope.
In a concrete refinement, a first detection channel could exhibit detection light which is emitted on the side of the object facing the light source, and a second detection channel could exhibit detection light which is emitted on the side of the object averted from the light source. Expressed more accurately, the first detection channel could exhibit reflected and/or fluorescent light. The second detection channel could, by contrast, exhibit transmitted and/or fluorescent light.
The detector assembly could be assigned to the first detection channel. In other words, it could be a detector assembly which is already present in the descanning arrangement in the case of a conventional microscope.
With regard to a concrete superimposition of two detection channels, the detection light of the second detection channel could be guided to the detector assembly by means of the superimposing device. This case could, in particular, concern an already present descanning detector assembly.
In an alternative refinement, both the detection light of the first detection channel and the detection light of the second detection channel could be guided to the detector assembly by means of the superimposing device. In this case, the detector assembly could be a detector assembly which is provided specifically for the superimposing technique and which is, if appropriate, implemented in addition to a descanning detector assembly already present. The detector assembly could be a nondescanning detector assembly, in particular.
With regard to effective optical superimposition of the detection channels, it could be possible to detect reflected light of the first detection channel and transmitted light of the second detection channel jointly, preferably in one and the same detector. Reflected light of the first detection channel and transmitted light of the second detection channel could in this case be approximately in the same wavelength region.
Furthermore, with reference to effective superimposition of the detection channels, fluorescent light of the first detection channel and fluorescent light of the second detection channel could be detected jointly, preferably in one and the same detector. Fluorescent light of the first detection channel and fluorescent light of the second detection channel could in this case be approximately in the same wavelength region.
Depending on the number of wavelength regions to be detected, it would be possible to provide a plurality of detectors which could then detect different wavelength regions.
The superimposing device could have a light-guiding device in order to ensure reliable superimposition of the detection channels. In a structurally particularly simple way, the light-guiding device could have an optical fiber. Glass fibers, in particular, can be used in this case. In particular, the light-guiding device could have an optical conductor filled with a liquid.
In an alternative refinement, the light-guiding device could have at least one mirror. One or more lenses could be assigned to the mirror or the mirrors. This renders it possible to construct mirror/lens arrangements which likewise permit reliable light guidance.
With regard to a particularly selective detection of the detection light, the detector assembly could be assigned a light-splitting device for splitting the detection light into different wavelength regions. The light-splitting device could have at least one color beam splitter in a particularly simple way.
As an alternative or addition hereto, the light-splitting device could have at least one partially transparent mirror. A bandpass or blocking filter could be arranged downstream of this mirror or these mirrors. In the case of the use of mirrors as splitting component, as well, it would also be possible to arrange a plurality of such mirrors in series, if appropriate with a downstream bandpass or blocking filter. Splitting the fluorescent light into a plurality of spectral regions is also possible thereby.
As an alternative to the use of color beam splitters or mirrors, it would be possible to use for splitting purposes a multiband detector. Splitting the fluorescent light into a plurality of spectral regions is also possible with the aid of such a multiband detector.
A laser could be used in a particularly advantageous way as light source. However, it is also conceivable to use other suitable light sources.